Generator-specific targets of mitochondrial reactive oxygen species

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for “signaling” and “damaging” ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc₁ complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H₂O₂) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.     

The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide

Neurodegenerative diseases, a subset of age-driven diseases, have been known to exhibit increased oxidative stress. The resultant increase in reactive oxygen species (ROS) has long been viewed as a detrimental byproduct of many cellular processes. Despite this, therapeutic approaches using antioxidants were deemed unsuccessful in circumventing neurodegenerative diseases. In recent times, it is widely accepted that these toxic by-products could act as secondary messengers, such as hydrogen peroxide (H₂O₂), to drive important signaling pathways. Notably, mitochondria are considered one of the major producers of ROS, especially in the production of mitochondrial H₂O₂. As a secondary messenger, cellular H₂O₂ can initiate redox signaling through oxidative post-translational modifications (oxPTMs) on the thiol group of the amino acid cysteine. With the current consensus that cellular ROS could drive important biological signaling pathways through redox signaling, researchers have started to investigate the role of cellular ROS in the pathogenesis of neurodegenerative diseases. Moreover, mitochondrial dysfunction has been linked to various neurodegenerative diseases, and recent studies have started to focus on the implications of mitochondrial ROS from dysfunctional mitochondria on the dysregulation of redox signaling. Henceforth, in this review, we will focus our attention on the redox signaling of mitochondrial ROS, particularly on mitochondrial H₂O₂, and its potential implications with neurodegenerative diseases.Subject terms: Post-translational modifications, Neurodegenerative diseases     

Oxygen and reactive oxygen species-dependent regulation of plant growth and development

Oxygen and reactive oxygen species (ROS) have been co-opted during evolution into the regulation of plant growth, development, and differentiation. ROS and oxidative signals arising from metabolism or phytohormone-mediated processes control almost every aspect of plant development from seed and bud dormancy, liberation of meristematic cells from the quiescent state, root and shoot growth, and architecture, to flowering and seed production. Moreover, the phytochrome and phytohormone-dependent transmissions of ROS waves are central to the systemic whole plant signaling pathways that integrate root and shoot growth. The sensing of oxygen availability through the PROTEOLYSIS 6 (PRT6) N-degron pathway functions alongside ROS production and signaling but how these pathways interact in developing organs remains poorly understood. Considerable progress has been made in our understanding of the nature of hydrogen peroxide sensors and the role of thiol-dependent signaling networks in the transmission of ROS signals. Reduction/oxidation (redox) changes in the glutathione (GSH) pool, glutaredoxins (GRXs), and thioredoxins (TRXs) are important in the control of growth mediated by phytohormone pathways. Although, it is clear that the redox states of proteins involved in plant growth and development are controlled by the NAD(P)H thioredoxin reductase (NTR)/TRX and reduced GSH/GRX systems of the cytosol, chloroplasts, mitochondria, and nucleus, we have only scratched the surface of this multilayered control and how redox-regulated processes interact with other cell signaling systems.

Oxygen and reactive oxygen species regulate plant growth, development, and differentiation through multiple interlinked signaling pathways.

Advances

• Developmentally regulated hypoxia and reactive oxygen species (ROS) production are key features of the stem cell niches, providing information about stem cell position, the environment, and metabolic state.
• Protein cysteine oxidation is central to oxygen and ROS signaling. However, S-nitrosylation, S-glutathionylation, S-sulfhydration, and S-sulfenylation modifications can occur on the same cysteine. The influence of each modification on stability, localization, and function remains unknown.
• Numerous intersecting ROS signaling pathways are probable and likely depend on the site of ROS production and the nature of the oxidized receptor protein. ROS sensors such as the hydrogen peroxide (H₂O₂)-INDUCED Ca²⁺ INCREASES 1 (HPCA1) leucine rich receptor kinase translate redox signals into protein modifications to regulate signaling cascades. H₂O₂ perception/transduction is dependent on thiol-dependent mechanisms policed by the ferredoxin/thioredoxin (TRX), NAD(P)H TRX reductase C (NTRC), reduced glutathione (GSH), and glutaredoxin (GRX) systems.
• ROS waves transmit redox signals from cell to cell in the apoplast, and probably through plasmodesmata. Long-distance transport of H₂O₂ and other ROS, therefore, appears to be unnecessary. Similarly, contact sites between organelles allow ROS transfer.
• Convergence points for oxygen and ROS signaling occur on proteins such as ROH OF PLANT 2 (ROP2) GTPase,RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), and TRX-h to regulate meristematic activity via TARGET OF RAPAMYCIN (TOR) kinase activity.

     

Nitration Transforms a Sensitive Peroxiredoxin 2 into a More Active and Robust Peroxidase

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H₂O₂-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H₂O₂ accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.     

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

During the past several years, major advances have been made in understanding how reactive oxygen species (ROS) and nitrogen species (RNS) participate in signal transduction. Identification of the specific targets and the chemical reactions involved still remains to be resolved with many of the signaling pathways in which the involvement of reactive species has been determined. Our understanding is that ROS and RNS have second messenger roles. While cysteine residues in the thiolate (ionized) form found in several classes of signaling proteins can be specific targets for reaction with H₂O₂ and RNS, better understanding of the chemistry, particularly kinetics, suggests that for many signaling events in which ROS and RNS participate, enzymatic catalysis is more likely to be involved than non-enzymatic reaction. Due to increased interest in how oxidation products, particularly lipid peroxidation products, also are involved with signaling, a review of signaling by 4-hydroxy-2-nonenal (HNE) is included. This article focuses on the chemistry of signaling by ROS, RNS, and HNE and will describe reactions with selected target proteins as representatives of the mechanisms rather attempt to comprehensively review the many signaling pathways in which the reactive species are involved.     

Regulation of reactive oxygen species generation in cell signaling

Reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H₂O₂) are thought to be byproducts of aerobic respiration with damaging effects on DNA, protein, and lipid. A growing body of evidence indicates, however, that ROS are involved in the maintenance of redox homeostasis and various cellular signaling pathways. ROS are generated from diverse sources including mitochondrial respiratory chain, enzymatic activation of cytochrome p450, and NADPH oxidases further suggesting involvement in a complex array of cellular processes. This review summarizes the production and function of ROS. In particular, how cytosolic and membrane proteins regulate ROS generation for intracellular redox signaling will be detailed.     

Integration of superoxide formation and cristae morphology for mitochondrial redox signaling

The mitochondrial network provides the central cell’s energetic and regulatory unit, which besides ATP and metabolite production participates in cellular signaling through regulated reactive oxygen species (ROS) production and various protein/ion fluxes. The inner membrane forms extensive folds, called cristae, i.e. cavities enfolded from and situated perpendicularly to its inner boundary membrane portion, which encompasses an inner cylinder within the outer membrane tubule. Mitochondrial cristae ultramorphology reflects various metabolic, physiological or pathological states. Since the mitochondrion is typically a predominant superoxide source and generated ROS also serve for the creation of information redox signals, we review known relationships between ROS generation within the respiratory chain complexes of cristae and cristae morphology. Notably, it is emphasized that cristae shape is governed by ATP-synthase dimers, MICOS complexes, OPA1 isoforms and the umbrella of their regulation, and also dependent on local protonmotive force (electrical potential component) in cristae. Cristae are also affected by redox-sensitive kinases/phosphatases or p66SHC. ATP-synthase dimers decrease in the inflated intracristal space, diminishing pH and hypothetically having minimal superoxide formation. Matrix-released signaling superoxide/H₂O₂ is predominantly integrated along mitochondrial tubules, whereas the diffusion of intracristal signaling ROS species is controlled by crista junctions, the widening of which enables specific retrograde redox signaling such as during hypoxic cell adaptation. Other physiological cases of H₂O₂ release from the mitochondrion include the modulation of insulin release in pancreatic β-cells, enhancement of insulin signaling in peripheral tissues, signaling by T-cell receptors, retrograde signaling during the cell cycle and cell differentiation, specifically that of adipocytes.     

Redox regulation of ER and mitochondrial Ca2+ signaling in cell survival and death

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca²⁺ signaling at the ER/mitochondrial interface – notably, inositol trisphosphate receptors (IP₃Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.     

Peroxisomes sense and respond to environmental cues by regulating ROS and RNS signalling networks

Background Peroxisomes are highly dynamic, metabolically active organelles that used to be regarded as a sink for H₂O₂ generated in different organelles. However, peroxisomes are now considered to have a more complex function, containing different metabolic pathways, and they are an important source of reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species (RNS). Over-accumulation of ROS and RNS can give rise oxidative and nitrosative stress, but when produced at low concentrations they can act as signalling molecules.Scope This review focuses on the production of ROS and RNS in peroxisomes and their regulation by antioxidants. ROS production is associated with metabolic pathways such as photorespiration and fatty acid β-oxidation, and disturbances in any of these processes can be perceived by the cell as an alarm that triggers defence responses. Genetic and pharmacological studies have shown that photorespiratory H₂O₂ can affect nuclear gene expression, regulating the response to pathogen infection and light intensity. Proteomic studies have shown that peroxisomal proteins are targets for oxidative modification, S-nitrosylation and nitration and have highlighted the importance of these modifications in regulating peroxisomal metabolism and signalling networks. The morphology, size, number and speed of movement of peroxisomes can also change in response to oxidative stress, meaning that an ROS/redox receptor is required. Information available on the production and detection of NO/RNS in peroxisomes is more limited. Peroxisomal homeostasis is critical for maintaining the cellular redox balance and is regulated by ROS, peroxisomal proteases and autophagic processes.Conclusions Peroxisomes play a key role in many aspects of plant development and acclimation to stress conditions. These organelles can sense ROS/redox changes in the cell and thus trigger rapid and specific responses to environmental cues involving changes in peroxisomal dynamics as well as ROS- and NO-dependent signalling networks, although the mechanisms involved have not yet been established. Peroxisomes can therefore be regarded as a highly important decision-making platform in the cell, where ROS and RNS play a determining role.     

Dopamine signaling impairs ROS modulation by mitochondrial hexokinase in human neural progenitor cells

Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D₁R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H₂O₂ and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.     

The antioxidant machinery of the endoplasmic reticulum: Protection and signaling

Cellular metabolism is inherently linked to the production of oxidizing by-products, including reactive oxygen species (ROS) hydrogen peroxide (H₂O₂). When present in excess, H₂O₂ can damage cellular biomolecules, but when produced in coordinated fashion, it typically serves as a mobile signaling messenger. It is therefore not surprising that cell health critically relies on both low-molecular-weight and enzymatic antioxidant components, which protect from ROS-mediated damage and shape the propagation and duration of ROS signals. This review focuses on H₂O₂–antioxidant cross talk in the endoplasmic reticulum (ER), which is intimately linked to the process of oxidative protein folding. ER-resident or ER-regulated sources of H₂O₂ and other ROS, which are subgrouped into constitutive and stimulated sources, are discussed and set into context with the diverse antioxidant mechanisms in the organelle. These include two types of peroxide-reducing enzymes, a high concentration of glutathione derived from the cytosol, and feedback-regulated thiol–disulfide switches, which negatively control the major ER oxidase ER oxidoreductin-1. Finally, new evidence highlighting emerging principles of H₂O₂-based cues at the ER will likely set a basis for establishing ER redox processes as a major line of future signaling research. A fundamental problem that remains to be solved is the specific, quantitative, time resolved, and targeted detection of H₂O₂ in the ER and in specialized ER subdomains.     

Signaling on the Stigma: Potential New Roles for ROS and NO in Plant Cell Signaling

Reactive oxygen species (ROS) and reactive nitrogen species, particularly NO, are key components of diverse signaling networks in animals and plants. We have recently shown that epidermal cells of stigmas from a range of different angiosperms accumulate relatively large amounts of ROS, principally H₂O₂, whereas pollen produces NO. Importantly, ROS/H₂O₂ levels appeared reduced in stigma cells supporting developing pollen grains compared to cells without pollen grains attached. To explore a possible link between pollen NO production and reduced levels of stigmatic ROS/H₂O₂, we supplied stigmas with NO and observed an overall reduction in levels of stigmatic ROS/H₂O₂. These new and unexpected data suggest a potential new signaling role for ROS/H₂O₂ and NO in pollen-stigma recognition processes.Key Words: stigma, pollen, reactive oxygen species, hydrogen peroxide, nitric oxide, signaling, defense     

Differential correlations between changes to glutathione redox state,protein ubiquitination,and stress-inducible HSPA chaperone expression after different types of oxidative stress

In primary bovine fibroblasts with an hspa1b/luciferase transgene, we examined the intensity of heat-shock response (HSR) following four types of oxidative stress or heat stress (HS), and its putative relationship with changes to different cell parameters, including reactive oxygen species (ROS), the redox status of the key molecules glutathione (GSH), NADP(H) NAD(H), and the post-translational protein modifications carbonylation, S-glutathionylation, and ubiquitination. We determined the sub-lethal condition generating the maximal luciferase activity and inducible HSPA protein level for treatments with hydrogen peroxide (H₂O₂), UVA-induced oxygen photo-activation, the superoxide-generating agent menadione (MN), and diamide (DA), an electrophilic and sulfhydryl reagent. The level of HSR induced by oxidative stress was the highest after DA and MN, followed by UVA and H₂O₂ treatments, and was not correlated to the level of ROS production nor to the extent of protein S-glutathionylation or carbonylation observed immediately after stress. We found a correlation following oxidative treatments between HSR and the level of GSH/GSSG immediately after stress, and the increase in protein ubiquitination during the recovery period. Conversely, HS treatment, which led to the highest HSR level, did not generate ROS nor modified or depended on GSH redox state. Furthermore, the level of protein ubiquitination was maximum immediately after HS and lower than after MN and DA treatments thereafter. In these cells, heat-induced HSR was therefore clearly different from oxidative stress-induced HSR, in which conversely early redox changes of the major cellular thiol predicted the level of HSR and polyubiquinated proteins.     

Beyond LDL oxidation: ROS in vascular signal transduction

The notion that oxidative stress contributes to the pathogenesis of vascular disease was originally driven by observations that low-density lipoprotein (LDL) modification is a prominent feature of atherosclerosis. More recently, it has become clear that the relation between oxidative stress and vascular disease goes beyond LDL oxidation and involves cellular production of reactive oxygen species (ROS). Considerable data now indicate that ROS represent an important means of cellular signaling, although the precise mechanisms whereby ROS accomplish this function remain unclear. Emerging data point to protein thiol groups as important targets for post-translational protein modification by ROS. In this review, the data linking ROS to cell signaling is discussed and the notion that ROS mediate a vascular "injury" response is proposed.     

Role of Metabolic H2O2 Generation: REDOX SIGNALING AND OXIDATIVE STRESS*

Hydrogen peroxide, the nonradical 2-electron reduction product of oxygen, is a normal aerobic metabolite occurring at about 10 nm intracellular concentration. In liver, it is produced at 50 nmol/min/g of tissue, which is about 2% of total oxygen uptake at steady state. Metabolically generated H₂O₂ emerged from recent research as a central hub in redox signaling and oxidative stress. Upon generation by major sources, the NADPH oxidases or Complex III of the mitochondrial respiratory chain, H₂O₂ is under sophisticated fine control of peroxiredoxins and glutathione peroxidases with their backup systems as well as by catalase. Of note, H₂O₂ is a second messenger in insulin signaling and in several growth factor-induced signaling cascades. H₂O₂ transport across membranes is facilitated by aquaporins, denoted as peroxiporins. Specialized protein cysteines operate as redox switches using H₂O₂ as thiol oxidant, making this reactive oxygen species essential for poising the set point of the redox proteome. Major processes including proliferation, differentiation, tissue repair, inflammation, circadian rhythm, and aging use this low molecular weight oxygen metabolite as signaling compound.